A molecular complex of Cav1.2/CaMKK2/CaMK1a in caveolae is responsible for vascular remodeling via excitation-transcription coupling.

Elevation of intracellular Ca2+ concentration ([Ca2+]i) activates Ca2+/calmodulin-dependent kinases (CaMK) and promotes gene transcription. This signaling pathway is referred to as excitation­transcription (E-T) coupling. Although vascular myocytes can exhibit E-T coupling, the molecular mechanisms and physiological/pathological roles are unknown. Multiscale analysis spanning from single molecules to whole organisms has revealed essential steps in mouse vascular myocyte E-T coupling. Upon a depolarizing stimulus, Ca2+ influx through Cav1.2 voltage-dependent Ca2+ channels activates CaMKK2 and CaMK1a, resulting in intranuclear CREB phosphorylation. Within caveolae, the formation of a molecular complex of Cav1.2/CaMKK2/CaMK1a is promoted in vascular myocytes. Live imaging using a genetically encoded Ca2+ indicator revealed direct activation of CaMKK2 by Ca2+ influx through Cav1.2 localized to caveolae. CaMK1a is phosphorylated by CaMKK2 at caveolae and translocated to the nucleus upon membrane depolarization. In addition, sustained depolarization of a mesenteric artery preparation induced genes related to chemotaxis, leukocyte adhesion, and inflammation, and these changes were reversed by inhibitors of Cav1.2, CaMKK2, and CaMK, or disruption of caveolae. In the context of pathophysiology, when the mesenteric artery was loaded by high pressure in vivo, we observed CREB phosphorylation in myocytes, macrophage accumulation at adventitia, and an increase in thickness and cross-sectional area of the tunica media. These changes were reduced in caveolin1-knockout mice or in mice treated with the CaMKK2 inhibitor STO609. In summary, E-T coupling depends on Cav1.2/CaMKK2/CaMK1a localized to caveolae, and this complex converts [Ca2+]i changes into gene transcription. This ultimately leads to macrophage accumulation and media remodeling for adaptation to increased circumferential stretch.

Melatonin inhibits voltage-gated potassium KV4.2 channels and negatively regulates melatonin secretion in rat pineal glands.

Melatonin is synthesized in and secreted from the pineal glands, and regulates circadian rhythms. Although melatonin has been reported to modulate the activity of ion channels in several tissues, its effects on pineal ion channels remain unclear. In the present study, the effects of melatonin on voltage-gated K+ (KV) channels, which play a role in regulating the resting membrane potential, were examined in rat pinealocytes. The application of melatonin reduced pineal KV currents in a concentration-dependent manner (IC50=309 mM). An expression analysis revealed that KV4.2 channels were highly expressed in rat pineal glands. Melatonin-sensitive currents were abolished by the small interfering RNA knockdown of KV4.2 channels in rat pinealocytes. In human embryonic kidney 293 (HEK293) cells expressing KV4.2 channels, melatonin decreased outward currents (IC50=479 mM). Inhibitory effects were mediated by a shift in voltage dependence from steady-state inactivation to a hyperpolarizing direction. This inhibition was observed even in the presence of 100 nM luzindole, an antagonist of melatonin receptors. Melatonin also blocked the activity of KV4.3, KV1.1, and KV1.5 channels in reconstituted HEK293 cells. The application of 1 mM melatonin caused membrane depolarization in rat pinealocytes. Furthermore, KV4.2 channel inhibition by 5 mM 4-aminopyridine attenuated melatonin secretion induced by 1 mM noradrenaline in rat pineal glands. These results strongly suggest that melatonin directly inhibited KV4.2 channels and caused membrane depolarization in pinealocytes, resulting in a decrease in melatonin secretion through parasympathetic signaling pathway. This mechanism may function as a negative-feedback mechanism of melatonin secretion in pineal glands.

Caveolin-1 forms a complex with P2X7 receptor and tunes P2X7-mediated ATP signaling in mouse bone marrow-derived macrophages.

The ionotropic purinergic P2X7 receptor responds to extracellular ATP and can trigger proinflammatory immune signaling in macrophages. Caveolin-1 (Cav-1) is known to modulate functions of macrophages and innate immunity. However, it is unknown how Cav-1 modulates P2X7 receptor activity in macrophages. We herein examined P2X7 receptor activity and macrophage functions using bone marrow-derived macrophages (BMDMs) from wild-type (WT) and Cav-1 knockout (KO) mice. ATP (1 mM) application caused biphasic increase in cytosolic [Ca2+] and sustained decrease in cytosolic [K+]. A specific P2X7 receptor blocker, A-740003, inhibited the maintained cytosolic [Ca2+] increase and cytosolic [K+] decrease. Total internal reflection fluorescent imaging and proximity ligation assays revealed a novel molecular complex formation between P2X7 receptors and Cav-1 in WT BMDMs that were stimulated with lipopolysaccharides. This molecular coupling was increased by ATP application. Specifically, the ATP-induced Ca2+ influx and K+ efflux through P2X7 receptors were increased in Cav-1 KO BMDMs, even though the total and surface protein levels of P2X7 receptors in WT and Cav-1 KO BMDMs were unchanged. Cell-impermeable dye (TO-PRO3) uptake analysis revealed that macropore formation of P2X7 receptors was enhanced in Cav-1 KO BMDMs. Cav-1 KO BMDMs increased ATP-induced IL-1ß secretion, reactive oxygen species production, Gasdermin D (GSDMD) cleavage, and lactate dehydrogenase release indicating pyroptosis. A-740003 completely prevented ATP-induced pyroptosis. In combination, these datasets show that Cav-1 has a negative effect on P2X7 receptor activity in BMDMs and that Cav-1 in macrophages may contribute to finely tuned immune responses by preventing excessive IL-1ß secretion and pyroptosis.NEW & NOTEWORTHY In bone marrow-derived macrophages, Cav-1 suppresses the macropore formation of P2X7 receptors through their direct or indirect interactions, resulting in reduced membrane permeability of cations (Ca2+ and K+) and large cell-impermeable dye (TO-PRO3) induced by ATP. Cav-1 also inhibits ATP-induced IL-1ß secretion, ROS production, GSDMD cleavage, and pyroptosis. Cav-1 contributes to the maintenance of proper immune responses by finely tuning IL-1ß secretion and cell death in macrophages.

Mitofusin 1 and 2 differentially regulate mitochondrial function underlying Ca2+ signaling and proliferation in rat aortic smooth muscle cells.

Mitochondria play a substantial role in cytosolic Ca2+ buffering and energy metabolism. We recently demonstrated that mitofusin 2 (Mfn2) regulated Ca2+ signaling by tethering mitochondria and sarcoplasmic reticulum (SR), and thus, facilitated mitochondrial function and the proliferation of vascular smooth muscle cells (VSMCs). However, the physiological role of mitofusin 1 (Mfn1) on Ca2+ signaling and mitochondrial function remains unclear. Herein, the roles of Mfn1 and Mfn2 in mitochondrial function underlying Ca2+ signaling, ATP production, and cell proliferation were examined in rat aortic smooth muscle A10 cells. Following an arginine vasopressin-induced increase in cytosolic Ca2+ concentration ([Ca2+]cyt), Mfn2 siRNA (siMfn2) reduced cytosolic Ca2+ removal and mitochondrial Ca2+ uptake. However, Mfn1 siRNA (siMfn1) attenuated mitochondrial Ca2+ uptake, facilitated Ca2+ removal from mitochondria, and resulted in increased [Ca2+]cyt, which was mediated by the downregulation of mitochondrial Ca2+ uniporter (MCU) expression and the upregulation of mitochondrial Na+/Ca2+ exchanger (NCLX) expression. Furthermore, siMfn1 increased the mitochondrial membrane potential, ATP production by adenine nucleotide translocase (ANT), and cell proliferation, whereas siMfn2 exhibited the opposite responses. In conclusion, Mfn1 modulates the expressions of MCU, NCLX, and ANT, and Mfn2 tethers mitochondria to SR, which demonstrates their different mitochondrial functions for Ca2+ signaling, ATP production, and the proliferation of VSMCs.

Pimaric acid reduces vasoconstriction via BKCa channel activation and VDCC inhibition in rat pulmonary arterial smooth muscles.

Pulmonary vessels play a pivotal role in oxygen circulation. We previously demonstrated that pimaric acid (PiMA) activated large-conductance Ca2+-activated K+ (BKCa) channels and inhibited voltage-dependent Ca2+ channels (VDCCs). In the present study, PiMA attenuated vasoconstriction induced by high K+ or endothelin-1 in rat pulmonary arterial smooth muscles (PASMs). PiMA also reduced high K+-induced cytosolic [Ca2+] increase in PASM cells. PiMA increased BKCa currents and decreased VDCC currents. BKCa channels and VDCCs were formed by the α/ß1 and α1C/α1D/ß2/ß3 subunits, respectively. These results indicate that PiMA induces vasorelaxation through the dual effects of BKCa channel activation and VDCC inhibition in PASMs.

Mitofusin 2 positively regulates Ca2+ signaling by tethering the sarcoplasmic reticulum and mitochondria in rat aortic smooth muscle cells.

Mitochondria buffer cytosolic Ca2+ increases following Ca2+ influx from extracellular spaces, and Ca2+ release from intracellular Ca2+ store sites under physiological circumstances. Therefore, close contact of mitochondria with the sarcoplasmic reticulum (SR) is required for maintaining Ca2+ homeostasis. Mitofusin 2 (Mfn2) localizes in both mitochondrial and SR membranes and is hypothesized to optimize the distance and Ca2+ transfer between these organelles. However, the physiological significance of Mfn2 in vascular smooth muscle cells (VSMCs) is poorly understood. In the present study, the role of Mfn2 in the physical and functional couplings between SR and mitochondria was examined in rat aortic smooth muscle cells (rASMCs) by confocal and electron microscope imaging. When Mfn2 was knocked down using siRNA in rASMCs, the mean distance between these organelles was extended from 16.2 to 21.6 nm. The increase in the cytosolic Ca2+ concentration ([Ca2+]cyt) induced by 100 nM arginine vasopressin (AVP) was not affected by Mfn2 siRNA knockdown, whereas cytosolic Ca2+ removal was slower after Mfn2 knockdown. Following the AVP-induced [Ca2+]cyt increase, mitochondrial Ca2+ uptake and Ca2+ refill into the SR were attenuated by Mfn2 knockdown. In addition, Mfn2-knockdown cells exhibited a loss of mitochondrial membrane potential (ΔΨmito) and lower ATP levels in mitochondria. Moreover, Mfn2 knockdown inhibited cell proliferation. In contrast, Mfn2 overexpression increased ΔΨmito and cell growth. This study strongly suggests that Mfn2 is responsible for SR-mitochondria Ca2+ signaling by tethering mitochondria to SR, thereby regulating ATP production and proliferation of VSMCs.

Involvement of small-conductance Ca2+-activated K+ (SKCa2) channels in spontaneous Ca2+ oscillations in rat pinealocytes.

Melatonin secretion from the pineal glands regulates circadian rhythms in mammals. Melatonin production is decreased by an increase in cytosolic Ca2+ concentration following the activation of nicotinic acetylcholine receptors in parasympathetic systems. We previously reported that pineal Ca2+ oscillations were regulated by voltage-dependent Ca2+ channels and large-conductance Ca2+-activated K+ (BKCa) channels, which inhibited melatonin production. In the present study, the contribution of small- and intermediate-conductance Ca2+-activated K+ (SKCa and IKCa) channels to the regulation of spontaneous Ca2+ oscillations was examined in rat pinealocytes. The amplitude and frequency of spontaneous Ca2+ oscillations were increased by a SKCa channel blocker (100 nM apamin), but not by an IKCa channel blocker (1 µM TRAM-34). On the other hand, they were decreased by a SKCa channel opener (100 µM DCEBIO), but not by an IKCa channel opener (1 µM DCEBIO). Expression analyses using quantitative real-time PCR, immunocytochemical staining, and Western blotting revealed that the SKCa2 channel subtype was abundantly expressed in rat pinealocytes. Moreover, the enhanced amplitude of Ca2+ oscillations in the presence of apamin was further increased by a BKCa channel blocker (1 µM paxilline). These results suggest that the activity of SKCa2 channels regulates cytosolic Ca2+ signaling and melatonin production during parasympathetic activation in pineal glands.

Reciprocal Relationship between Ca2+ Signaling and Ca2+-Gated Ion Channels as a Potential Target for Drug Discovery.

Cellular Ca2+ signaling functions as one of the most common second messengers of various signal transduction pathways in cells and mediates a number of physiological roles in a cell-type dependent manner. Ca2+ signaling also regulates more general and fundamental cellular activities, including cell proliferation and apoptosis. Among ion channels, Ca2+-permeable channels in the plasma membrane as well as endo- and sarcoplasmic reticulum membranes play important roles in Ca2+ signaling by directly contributing to the influx of Ca2+ from extracellular spaces or its release from storage sites, respectively. Furthermore, Ca2+-gated ion channels in the plasma membrane often crosstalk reciprocally with Ca2+ signals and are central to the regulation of cellular functions. This review focuses on the physiological and pharmacological impact of i) Ca2+-gated ion channels as an apparatus for the conversion of cellular Ca2+ signals to intercellularly propagative electrical signals and ii) the opposite feedback regulation of Ca2+ signaling by Ca2+-gated ion channel activities in excitable and non-excitable cells.

Local Ca2+ Signals within Caveolae Cause Nuclear Translocation of CaMK1α in Mouse Vascular Smooth Muscle Cells.

An increase in intracellular Ca2+ concentration ([Ca2+]i) activates Ca2+-sensitive enzymes such as Ca2+/calmodulin-dependent kinases (CaMK) and induces gene transcription in various types of cells. This signaling pathway is called excitation-transcription (E-T) coupling. Recently, we have revealed that a L-type Ca2+ channel/CaMK kinase (CaMKK) 2/CaMK1α complex located within caveolae in vascular smooth muscle cells (SMCs) can convert [Ca2+]i changes to gene transcription profiles that are related to chemotaxis. Although CaMK1α is expected to be the key molecular identity that can transport Ca2+ signals originated within caveolae to the nucleus, data sets directly proving this scheme are lacking. In this study, multicolor fluorescence imaging methods were utilized to address this question. Live cell imaging using mouse primary aortic SMCs revealed that CaMK1α can translocate from the cytosol to the nucleus; and that this movement was blocked by nifedipine or a CaMKK inhibitor, STO609. Experiments using two types of Ca2+ chelators, ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA) and 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), combined with caveolin-1 knockout (cav1-KO) mice showed that local Ca2+ events within caveolae are required to trigger this CaMK1α nuclear translocation. Importantly, overexpression of cav1 in isolated cav1-KO myocytes recovered the CaMK1α translocation. In SMCs freshly isolated from mesenteric arteries, CaMK1α was localized mainly within caveolae in the resting state. Membrane depolarization induced both nuclear translocation and phosphorylation of CaMK1α. These responses were inhibited by nifedipine, STO609, cav1-KO, or BAPTA. These new findings strongly suggest that CaMK1α can transduce Ca2+ signaling generated within or very near caveolae to the nucleus and thus, promote E-T coupling.

Swelling-activated ClC-3 activity regulates prostaglandin E2 release in human OUMS-27 chondrocytes.

Articular chondrocytes are exposed to dynamic osmotic environments during normal joint loading, and thus, require effective volume regulatory mechanisms. A regulatory volume decrease (RVD) is one of the mechanisms for protecting chondrocytes from swelling and damage. Swelling-activated Cl- currents (ICl,swell) are responsible for the RVD, but the molecular identity in chondrocytes is largely unknown. In this study, we reveal that in human OUMS-27 chondrocytes, ICl,swell can be elicited by hypoosmotic stimulation (180 mOsm) and be inhibited by classical Cl- channel blockers, 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS) and niflumic acid, and be attenuated by siRNA knockdown of ClC-3. Our molecular analyses revealed that ClC-3A is expressed as a major splice variant in both human articular chondrocytes and OUMS-27 cells. The onset and early phase of RVD following hypoosmotic stress in OUMS-27 cells were affected by DIDS and ClC-3 knockdown. Hypoosmotic stimulation caused Ca2+ influx and subsequent release of prostaglandin E2 (PGE2) in OUMS-27 cells, and both of these responses were reduced by DIDS and ClC-3 knockdown. These results strongly suggest that ClC-3 is responsible for ICl,swell and RVD under the hypoosmotic environments. It is likely that ClC-3 is associated with the pathogenesis of cartilage degenerative diseases including osteoarthritis via PGE2 release.

Hypoxia increases the proliferation of brain capillary endothelial cells via upregulation of TMEM16A Ca2+-activated Cl- channels.

The blood-brain barrier (BBB) is mainly formed by brain capillary endothelial cells (BCECs) and is exposed to hypoxic environments under pathological conditions. The effects of hypoxia on the expression and activity of Ca2+-activated Cl- (ClCa) channels, TMEM16A, were examined in bovine brain endothelial t-BBEC117 cells and mouse BCECs. The expression of TMEM16A was upregulated by hypoxia. Whole-cell ClCa currents increased under hypoxia. Hypoxia also increased cell proliferation and trans-endothelial permeability, which were attenuated by ClCa channel blockers or TMEM16A siRNA. These findings are useful for elucidating the pathological role of TMEM16A ClCa channels in the BBB during cerebral ischemia.

TMEM16A Ca2+-Activated Cl- Channel Regulates the Proliferation and Migration of Brain Capillary Endothelial Cells.

The blood-brain barrier (BBB) is essential for the maintenance of homeostasis in the brain. Brain capillary endothelial cells (BCECs) comprise the BBB, and thus a delicate balance between their proliferation and death is required. Although the activity of ion channels in BCECs is involved in BBB functions, the underlying molecular mechanisms remain unclear. In the present study, the molecular components of Ca2+-activated Cl- (ClCa) channels and their physiological roles were examined using mouse BCECs (mBCECs) and a cell line derived from bovine BCECs, t-BBEC117. Expression analyses revealed that TMEM16A was strongly expressed in mBCECs and t-BBEC117 cells. In t-BBEC117 cells, whole-cell Cl- currents were sensitive to the ClCa channel blockers, 100 µM niflumic acid and 10 µM T16Ainh-A01, and were also reduced markedly by small-interfering RNA (siRNA) knockdown of TMEM16A. Importantly, block of ClCa currents with ClCa channel blockers or TMEM16A siRNA induced membrane hyperpolarization. Moreover, treatment with TMEM16A siRNA caused an increase in resting cytosolic Ca2+ concentration ([Ca2+]cyt). T16Ainh-A01 reduced cell viability in a concentration-dependent manner. Either ClCa channel blockers or TMEM16A siRNA also curtailed cell proliferation and migration. Furthermore, ClCa channel blockers attenuated the trans-endothelial permeability. In combination, these results strongly suggest that TMEM16A contributes to ClCa channel conductance and can regulate both the resting membrane potential and [Ca2+]cyt in BCECs. Our data also reveal how these BCECs may be involved in the maintenance of BBB functions, as both the proliferation and migration are altered following changes in channel activity. SIGNIFICANCE STATEMENT: In brain capillary endothelial cells (BCECs) of the blood-brain barrier (BBB), TMEM16A is responsible for Ca2+-activated Cl- channels and can regulate both the resting membrane potential and cytosolic Ca2+ concentration, contributing to the proliferation and migration of BCECs. The present study provides novel information on the molecular mechanisms underlying the physiological functions of BCECs in the BBB and a novel target for therapeutic drugs for disorders associated with dysfunctions in the BBB.

A junctophilin-caveolin interaction enables efficient coupling between ryanodine receptors and BKCa channels in the Ca2+ microdomain of vascular smooth muscle.

Functional coupling between large-conductance Ca2+-activated K+ (BKCa) channels in the plasma membrane (PM) and ryanodine receptors (RyRs) in the sarcoplasmic reticulum (SR) is an essential mechanism for regulating mechanical force in most smooth muscle (SM) tissues. Spontaneous Ca2+ release through RyRs (Ca2+ sparks) and subsequent BKCa channel activation occur within the PM-SR junctional sites. We report here that a molecular interaction of caveolin-1 (Cav1), a caveola-forming protein, with junctophilin-2 (JP2), a bridging protein between PM and SR, positions BKCa channels near RyRs in SM cells (SMCs) and thereby contributes to the formation of a molecular complex essential for Ca2+ microdomain function. Approximately half of all Ca2+ sparks occurred within a close distance (<400 nm) from fluorescently labeled JP2 or Cav1 particles, when they were moderately expressed in primary SMCs from mouse mesenteric artery. The removal of caveolae by genetic Cav1 ablation or methyl-ß-cyclodextrin treatments significantly reduced coupling efficiency between Ca2+ sparks and BKCa channel activity in SMCs, an effect also observed after JP2 knockdown in SMCs. A 20-amino acid-long region in JP2 appeared to be essential for the observed JP2-Cav1 interaction, and we also observed an interaction between JP2 and the BKCa channel. It can be concluded that the JP2-Cav1 interaction provides a structural and functional basis for the Ca2+ microdomain at PM-SR junctions and mediates cross-talk between RyRs and BKCa channels, converts local Ca2+ sparks into membrane hyperpolarization, and contributes to stabilizing resting tone in SMCs.

Roles of LRRC26 as an auxiliary γ1-subunit of large-conductance Ca2+-activated K+ channels in bronchial smooth muscle cells.

In visceral smooth muscle cells (SMCs), the large-conductance Ca2+-activated K+ (BK) channel is one of the key elements underlying a negative feedback mechanism that is essential for the regulation of intracellular Ca2+ concentration. Although leucine-rich repeat-containing (LRRC) proteins have been identified as novel auxiliary γ-subunits of the BK channel (BKγ) in several cell types, its physiological roles in SMCs are unclear. The BKγ expression patterns in selected SM tissues were examined using real-time PCR analyses and Western blotting. The functional contribution of BKγ1 to BK channel activity was examined by whole cell patch-clamp in SMCs and heterologous expression systems. BKγ1 expression in mouse bronchial SMCs (mBSMCs) was higher than in other several SMC types. Coimmunoprecipitation and total internal reflection fluorescence imaging analyses revealed molecular interaction between BKα and BKγ1 in mBSMCs. Under voltage-clamp, steady-state activation of BK channel currents at pCa 8.0 in mBSMCs occurred in a voltage range comparable to that of reconstituted BKα/BKγ1 complex. However, this range was much more negative than in mouse aortic SMCs (mASMCs) or in HEK293 cells expressing BKα alone and ß-subunit (BKß1). Mallotoxin, a selective activator of BK channel that lacks BKγ1, dose-dependently activated BK currents in mASMCs but not in mBSMCs. The abundant expression of BKγ1 in mBSMCs extensively facilitates BK channel activity to keep the resting membrane potential at negative values and prevents contraction under physiological conditions.

Involvement of the γ1 subunit of the large-conductance Ca2+-activated K+ channel in the proliferation of human somatostatinoma cells.

Pancreatic neuroendocrine tumors (pNETs) occur due to the abnormal growth of pancreatic islet cells and predominantly develop in the duodenal-pancreatic region. Somatostatinoma is one of the pNETs associated with tumors of pancreatic δ cells, which produce and secrete somatostatin. Limited information is currently available on the pathogenic mechanisms of somatostatinoma. The large-conductance Ca2+-activated K+ (BKCa) channel is expressed in several types of cancer cells and regulates cell proliferation, migration, invasion, and metastasis. In the present study, the functional expression of the BKCa channel was examined in a human somatostatinoma QGP-1 cell line. In QGP-1 cells, outward currents were elicited by membrane depolarization at pCa 6.5 (300 nM) in the pipette solution and inhibited by the specific BKCa channel blocker, paxilline. Paxilline-sensitive currents were detected, even at pCa 8.0 (10 nM) in the pipette solution, in QGP-1 cells. In addition to the α and ß2-4 subunits of the BKCa channel, the novel regulatory γ1 subunit (BKCaγ1) was co-localized with the α subunit in QGP-1 cells. Paxilline-sensitive currents at pCa 8.0 in the pipette solution were reduced by the siRNA knockdown of BKCaγ1. Store-operated Ca2+ entry was smaller in BKCaγ1 siRNA-treated QGP-1 cells. The proliferation of QGP-1 cells was attenuated by paxilline or the siRNA knockdown of BKCaγ1. These results strongly suggest that BKCaγ1 facilitates the proliferation of human somatostatinoma cells. Therefore, BKCaγ1 may be a novel therapeutic target for somatostatinoma.

Oxidative stress facilitates cell death by inhibiting Orai1-mediated Ca2+ entry in brain capillary endothelial cells.

Brain capillary endothelial cells (BCECs) form the blood-brain barrier (BBB) and play an essential role in the regulation of its functions. Oxidative stress accumulates excessive reactive oxygen species (ROS) and facilitates the death of BCECs, leading to a dysfunctional BBB. However, the mechanisms underlying the death of BCECs under oxidative stress remain unclear. In the present study, the effects of oxidative stress on cell viability, ROS production, intracellular Ca2+ concentration, and protein expression were examined using a cell line derived from bovine BCECs, t-BBEC117. When t-BBEC117 cells were exposed to oxidative stress induced by hydrogen peroxide (H2O2, 10-100 µM), cell growth was inhibited in a dose-dependent manner. Oxidative stress by 30 µM H2O2 increased the production of ROS and its effects were blocked by the ROS scavenger, 10 mM N-acetyl-l-cysteine (NAC). In addition, oxidative stress reduced store-operated Ca2+ entry (SOCE) and this decrease was recovered by NAC or the Orai channel activator, 5 µM 2-aminoethyl diphenylborinate (2-APB). The siRNA knockdown of Orai1 revealed that Orai1 was mainly responsible for SOCE channels and its activity was decreased by oxidative stress. However, the protein expression of Orai1 and STIM1 was not affected by oxidative stress. Oxidative stress-induced cell death was rescued by 2-APB, NAC, or the STIM-Orai activating region. In conclusion, oxidative stress reduces Orai1-mediated SOCE and, thus, facilitates the death of BCECs.

Single Molecule Fluorescence Imaging Reveals the Stoichiometry of BKγ1 Subunit in Living HEK293 Cell Expression System.

Large conductance Ca2+-activated K+ (BKCa) channels are ubiquitously expressed in plasma membrane of both excitable and non-excitable cells and possess significant physiological functions. A tetrameric complex of α subunit (BKα) forms a functional pore of BKCa channel. The properties of BKCa channel, such as voltage-dependence, Ca2+ sensitivity and pharmacological responses, are extensively modulated by co-expressing accessory ß subunits (BKß), which can associate with BKα in one to one manner. Although the functional significance of newly identified γ subunits (BKγ) has been revealed, the stoichiometry between BKα and BKγ1 remains unclear. In the present study, we utilized a single molecule fluorescence imaging with a total internal reflection fluorescence (TIRF) microscope to directly count the number of green fluorescent protein (GFP)-tagged BKγ1 (BKγ1-GFP) within a single BKCa channel complex in HEK293 cell expression system. BKγ1-GFP significantly enhanced the BK channel activity even when the intracellular Ca2+ concentration was kept lower, i.e., 10 nM, than the physiological resting level. BKγ1-GFP stably formed molecular complexes with BKα-mCherry in the plasma membrane. Counting of GFP bleaching steps revealed that a BKCa channel can contain up to four BKγ1 per channel at the maximum. These results suggest that BKγ1 forms a BKCa channel complex with BKα in a 1 : 1 stoichiometry in a human cell line.

TMEM16A and TMEM16B channel proteins generate Ca2+-activated Cl- current and regulate melatonin secretion in rat pineal glands.

Pinealocytes regulate circadian rhythm by synthesizing and secreting melatonin. These cells generate action potentials; however, the contribution of specific ion channels to melatonin secretion from pinealocytes remains unclear. In this study, the involvement and molecular identity of Ca2+-activated Cl- (ClCa) channels in the regulation of melatonin secretion were examined in rat pineal glands. Treatment with the ClCa channel blockers, niflumic acid or T16Ainh-A01, significantly reduced melatonin secretion in pineal glands. After pineal K+ currents were totally blocked under whole-cell patch clamp conditions, depolarization and subsequent repolarization induced a slowly activating outward current and a substantial inward tail current, respectively. Both of these current changes were dependent on intracellular Ca2+ concentration and inhibited by niflumic acid and T16Ainh-A01. Quantitative real-time PCR, Western blotting, and immunocytochemical analyses revealed that TMEM16A and TMEM16B were highly expressed in pineal glands. siRNA knockdown of TMEM16A and/or TMEM16B showed that both channels contribute to ClCa currents in pinealocytes. Conversely, co-expression of TMEM16A and TMEM16B channels or the expression of this tandem channel in HEK293 cells mimicked the electrophysiological characteristics of ClCa currents in pinealocytes. Moreover, bimolecular fluorescence complementation, FRET, and co-immunoprecipitation experiments suggested that TMEM16A and TMEM16B can form heteromeric channels, as well as homomeric channels. In conclusion, pineal ClCa channels are composed of TMEM16A and TMEM16B subunits, and these fluxes regulate melatonin secretion in pineal glands.

Rapid Na+ accumulation by a sustained action potential impairs mitochondria function and induces apoptosis in HEK293 cells expressing non-inactivating Na+ channels.

The mechanisms underlying neuronal cell death induced by the rise of intracellular Na+ concentration ([Na+]i) following abnormal hyperexcitation are not fully understood. Previously, we have established a recombinant cell line derived from HEK293 cells, in which the occurrence of a sustained action potential (AP) induces cell death. Mutated voltage-gated Nav1.5 channel (IFM/QQQ) lacking inactivation, and inward rectifying K+ channel (Kir2.1) were co-expressed in HEK293 cells (IFM/QQQ + Kir2.1 cells). In this cell line, the rise of [Na+]i due to a sustained AP reached maximum within 15 min without concomitant [Ca2+]i rise, and then elicited significant externalization of phosphatidylserine and enhancement of caspase activity. Marked decreases in mitochondrial transmembrane potential and ATP concentration were also detected. The significant cell death occurred at 3 h from the AP onset and reached a steady state at around 12 h. The significant release of lactate dehydrogenase was not detected even after 12 h. These results provide novel findings that Na+ accumulation or/and possibly concomitant K+ loss elicits apoptosis presumably due to the mitochondrial dysfunction, which is attributable to neither the membrane depolarization nor [Ca2+]i change. This apoptotic mechanism may be involved, at least in part, in neuronal cell death under pathophysiological settings with abnormal hyperexcitability.

Conversion of Ca2+ oscillation into propagative electrical signals by Ca2+-activated ion channels and connexin as a reconstituted Ca2+ clock model for the pacemaker activity.

Conversion of intracellular Ca2+ signals to electrical activity results in multiple and differing physiological impacts depending on cell types. In some organs such as gastrointestinal and urinary systems, spontaneous Ca2+ oscillation in pacermaker cells can function essentially as a Ca2+ clock mechanism, which has been originally found in pacemaking in sinoatrial node cell of the heart. The conversion of discrete Ca2+ clock events to spontaneous electrical activity is an essential step for the initiation and propagation of pacemaker activity through the multicellular organs resulting in synchronized physiological functions. Here, a model of intracellular signal transduction from a Ca2+ oscillation to initiation of electrical slow waves and their propagation were reconstituted in HEK293 cells. This was accomplished based on ryanodine receptor (RyR) type 3, Ca2+-activated ion channels, i.e. small conductance Ca2+-activated K+ channel (SK2) or Ca2+-activated Cl- channel (TMEM16A), and connexin43 being heterologously co-expressed. The propagation of electrical waves was abolished or substantially reduced by treatment with selective blockers of the expressed channels and 18ß-glycyrrhetinic acid, a gap junction inhibitor, respectively. Thus, we demonstrated that the conversion of Ca2+ oscillation to electrical signals with cell to cell propagation can be reconstituted as a model of Ca2+ clock pacemaker activity by combinational expression of critical elements in heterologous expression system.